Conformational Difference in Human IgG2 Disulfide Isoforms Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Both recombinant and natural human IgG2 antibodies have several different disulfide bond isoforms, which possess different global structures, thermal stabilities, and biological activities. A detailed mapping of the structural difference among IgG2 disulfide isoforms, however, has not been established. In this work, we employed hydrogen/deuterium exchange mass spectrometry to study the conformation of three major IgG2 disulfide isoforms known as IgG2-B, IgG2-A1, and IgG2-A2 in two recombinant human IgG2 monoclonal antibodies. By comparing the protection factors between amino acid residues in isoforms B and A1 (the classical form), we successfully identified several local regions in which the IgG2-B isoform showed more solvent protection than the IgG2-A1 isoform. On the basis of three-dimensional structural models of IgG2, these identified regions were located on the Fab domains, close to the hinge, centered on the side where the two Fab arms faced each other in spatial proximity. We speculated that in the more solvent-protected B isoform, the two Fab arms were brought into contact by the nonclassical disulfide bonds, resulting in a more compact global structure. Loss of Fab domain flexibility in IgG2-B could limit its ability to access cell-surface epitopes, leading to reduced antigen binding potency. The A2 isoform was previously found to have disulfide linkages similar to those of the classical A1 isoform, but with different biophysical behaviors. Our data indicated that, compared to IgG2-A1, IgG2-A2 had less solvent protection in some heavy-chain Fab regions close the hinge, suggesting that the A2 isoform had more flexible Fab domains.

Alleviating nonlinear behavior of disulfide isoforms in the reversed-phase liquid chromatography of IgG2

“…These results reveal that the A1 form is not stable under the conditions of the chromatographic separation. This is consistent with a recent report that the A1 form is the least stable IgG2 isoform due to less protection of the disulfide bond [26]. These data in Figure2a and 2b support a conclusion that the conditions of reversed phase chromatography are capable of changing the sample composition for a sufficiently high concentration of the A1 isoform, but not for IgG2 concentrations typically used.…”

Evidence of disulfide bond scrambling during production of an antibody-drug conjugate

“…Detection of these species after disruption of all hinge disulfides indicates the presence of strong noncovalent forces between the HCs, in alignment with the extensive Fab-Fab and Fab-C H 2 interactions previously reported for the B isoform. 26,51 Our observed disulfide isoform conversion directionality is in agreement with previous studies that have reported conversion of the A → A/B → B forms in a reducing environment similar to physiological conditions. 24,25 The higher thermal stability of the IgG2-B mAb and ADCs observed in this study provides further evidence that IgG2 disulfide scrambling favors the thermodynamic product.…”

“…21,22 The higher-order structural differences at the Fab/C H 2 interface of the IgG2 isoforms affect the solvent accessibilities of the interchain disulfides. 26,27 One consideration for the ADC manufacturing process is the potential for disulfide bond rearrangement, which can occur at the preferred pH range for the partial reduction and conjugation reactions. [28][29][30][31][32] The rate of intramolecular thiol-disulfide exchange is accelerated when the conditions favor the degradation of disulfides and the formation of the thiolate anion, such as under the presence of a reducing reagent or in neutral or alkaline environments.…”

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

“…Potential approaches that capture this information include NMR, 13 EPR, 14 single molecule spectroscopy, 15,16 ion mobility mass spectrometry (IM-MS) 17 and hydrogen/deuterium (H/D) exchange mass spectrometry. 18 However, at present these approaches are not in routine use due to significant technical complexity and feasibility, instrument expense, time of assay, complex sample preparation and need for specialist analysis. Instead, a breadth of lower resolution approaches such as far-UV circular dichroism (CD), used to detect changes in protein secondary structure, and light scattering or size exclusion chromatography (SEC), are used to detect aggregation.…”